Recently, gene editing systems such as zinc finger nucleases, CRISPR/Cas systems, transcription activator-like effector nucleases (TALENs) and meganucleases have emerged as tools for the regulation of genes.
Monomeric Cas9 nuclease-based systems are directed to cleave specific DNA sequences by an associated ˜100-nt single RNA comprising 17-20 nucleotides that target the Cas9 nuclease to a site of interest (targeting RNA) by hybridizing to the target DNA site. While the simplicity of designing the targeting RNA to recognize predetermined sequences of DNA makes the CRISPR/Cas-based gene editing systems powerful, the large size of the Cas9 enzyme makes it nearly impossible to incorporate the system into a single vector for gene delivery in humans, and the sequence requirements of the Cas9 enzyme itself (e.g., PAM sequence) limit the sites to which it can be targeted.
In contrast, Zinc finger nucelase and TALEN-based gene editing system use much smaller enzyme components, such as the FokI nuclease, and the nucleases do not require specific binding sites. However, these systems are hampered by the use of protein-based DNA-targeting domains, which are difficult to engineer to bind specific DNA target sequences.
There is thus a need for a gene editing system which can be guided to a target DNA binding site using an easy-to-engineer component such as a targeting RNA but which utilizes a compact gene editing enzyme with no required sequence specificity.